Proton-translocating retinal protein

ABSTRACT

The invention relates to proton-translocating retinal proteins which exhibit a photocycle which is retarded as compared with the wild type and whose all-trans retinal contents in the light-adapted and dark-adapted states do not differ from each other by more than 10%. The invention furthermore relates to a photochromic composition and to the use of the proton-translocating retinal proteins and the photochromic composition.

[0001] The invention relates to a protein-translocating retinal protein,to a photochromic composition which comprises the proton-translocatingretinal protein and to the use of the proton-translocating retinalprotein and the composition.

[0002] Halobacteria are Archaea, which, alongside the Bacteria andEukarya, form a third domain of life. Archaea owe their name to theunusual biotopes in which they are found. They frequently live under“archaic” conditions, i.e. at extreme temperatures, salt concentrationsor pH values as may have prevailed on the early surface of the earth.

[0003] The halophilic Archaea comprise 9 genera (e.g. Halobacterium,Haloferax, Natronomonas, etc.) and are without exception extremophilic,i.e. they live in salt solutions whose concentrations extend from 2molar to saturation and which sometimes additionally exhibit alkaline pHvalues of up to pH 11. In nature, the Halobacteria are part of a complexecosystem. After the annual downpours, the salinity in salt-producinginstallations, which otherwise increases in association with permanentinsolation, or the conditions in the Dead Sea and other naturalhypersaline bodies of water, initially permit the photolithotrophicgrowth of the halotolerant green alga Duniella parva up to a saltcontent of about 12%. After the conditions which are optimal for it havebeen exceeded, Duniella dies and makes possible the massive growth ofhalophilic Archaea which, due to their carotenoid content, frequentlylead to these bodies of water becoming red in color.

[0004] Aside from fermentation and aerobic and anaerobic respiration,the halophilic Archaea possess an additional option, which is uniqueamong the Archaea, for converting energy: they are able to take up andconvert energy by means of retinal-dependent photosynthesis. In contrastto the green, chlorophyll-dependent photosynthesis, only one singleprotein is involved in the uptake and conversion of energy in this case,namely a light-driven proton-translocating retinal protein.

[0005] The best known example of such a proton-translocating retinalprotein is the archaebacterial proton pump bacteriorhodopsin (BR), whichuses light energy directly for generating an electrochemical protongradient which is converted into chemical energy. Bacteriorhodopsin isan intrinsic membrane protein having a molecular weight of approx. 26kDa. The polypeptide chain traverses the membrane seven times and inthis way forms a secondary structure consisting of seven helicaltransmembrane regions. A retinal (vitamin A aldehyde) is bondedcovalently to the side chain of a lysine belonging to the seventh helixin the interior of the protein. The resulting CH═N group is termed aSchiff's base (SB) and, in the initial state, is protonated on thenitrogen (SBH). The article by Haupts et al. (Annu. Rev. Biophys.Biomol. Struct. 28, 367-99, 1999) provides a review ofbacteriorhodopsin.

[0006] The BR chromophore absorbs yellow-green light maximally at 570nm, which means that BR appears violet to the human eye. After a photonhas been absorbed, the protein undergoes chemical and structural changeswhich lead to distinguishable, spectroscopically measurableintermediates. They are designated by the letters K, L, M, N and O andare in each case labeled with the wavelength at which absorption ismaximal. In a simplified manner, the cycle can be described as follows:

BR₅₇₀→K₅₉₀→L₅₅₀→M₄₁₀→N₅₆₀→O₆₄₀→BR₅₇₀

[0007] Bacteriorhodopsin is thus far the only retinal protein which isknown to occur in nature in the form of a two-dimensional crystal. Inthe bacterium, the crystals are located in what is known as the purplemembrane. The organization in the purple membrane stabilizes the proteinto such an extent that the protein has been proposed for a number oftechnical applications (summarized in Oesterhelt et al., Quarterly Rev.Biophysics 24, 425-478, 1991). These applications can make use of thechanges in the pH of the solution, in electrical potential and in colorwhich occur on illumination.

[0008] Thus, the change in color from the violet of thebacteriorhodopsin in the initial state BR₅₇₀ to the yellow in theintermediate state M₄₁₀ is the basis for applications in opticalinformation technology. The decomposition of the intermediate M₄₁₀,which has a lifetime of a few milliseconds, is the rate-determining stepin this cycle. The light intensity and the thermal decompositionconstant of the M intermediate determine the ratio of the colors violetand yellow in the photocycle.

[0009] In the dark, the retinylidene moiety of the bacteriorhodopsin ispresent as a mixture of all-trans, 15-anti and 13-cis, 15-synconfigurations in a ratio of about 60:40. Only the all-trans form of thechromophore mediates the physiological process of proton translocationand passes through the yellow intermediate M₄₁₀ (“trans cycle”). Whilethe absorption of photons in the 13-cis configuration also leads to acycle of color changes, i.e. the “cis cycle”, this latter differs fromthe “trans cycle” in that no yellow M-like intermediate is formed. Onillumination, the molecule jumps, with a very low degree of probability,from the “cis cycle” into the “trans cycle”. This change from the “ciscycle” to the “trans cycle” is termed light adaptation of thedark-adapted form. In practice this means that, after a sample has beenstored in the dark (dark adaptation), an initial illumination only leadsto about 60% of the theoretically possible M intermediate. Furtherillumination causes the sample to gradually adapt (light adaptation),such that finally all the molecules are transferred to the “trans cycle”and pass through the M intermediate.

[0010] The change of the molecules from the “cis cycle” to the “transcycle” leads to the absorption maximum being shifted by several nm andrepresents a substantial disadvantage for using bacteriorhodopsins whenproducing photochromic products, for example optical films or printinginks.

[0011] It would, therefore, be desirable to prepare proton-translocatingretinal proteins whose absorption maximum in the dark-adapted statecorresponded as precisely as possible to that in the light-adaptedstate. In addition to this, it would be advantageous if it were possibleto increase the stability of the M intermediate in order to be able toobserve the change in color, from violet to yellow, of the moleculespresent in the “trans cycle” as precisely as possible.

[0012] The invention now provides a proton-translocating retinal proteinwhich is selected from the group of:

[0013] (i) muteins of a natural proton-translocating retinal proteinderived from halophilic archaebacteria which exhibit a retardedphotocycle (type 1 mutation) and whose all-trans retinal contents in thelight-adapted and dark-adapted states do not differ from each other bymore than 10% (type 2 mutation) and/or

[0014] (ii) homologs of the muteins (i) which possess a retardedphotocycle and whose retinal isomer compositions in the light-adaptedand dark-adapted states do not differ from each other by more than 10%.

[0015] A mutein is understood as meaning protein-translocated retinalproteins which have been altered by a substitution, deletion orinsertion. Muteins can exhibit one or more mutations. In thisconnection, type 1 mutations are mutations which lead to a retardationof the photocycle as compared with the natural retinal protein derivedfrom Halobacterium salinarum (SEQ ID No. 1). In this context, thephotocycle is measured as described, for example, by Miller &Oesterhelt, Biochem. Biophys. Acta 1020, 57-64, 1990. Type 2 mutationslead to a constancy in the all-trans retinal content in thelight-adapted and dark-adapted protein which is greater than that of thenatural retinal protein derived from Halobacterium salinarum (SEQ ID No.1). In this context, the all-trans retinal content is determined asdescribed by Tittor et al., Biophys. J. 67, 1682-1690, 1994. Thepercentage value refers to the total content of retinal isomers whichcan be determined using the method mentioned. Normally, type 1 mutationsand type 2 mutations affect different amino acids. However, it is alsopossible for a single mutation to exhibit both the desired effects andtherefore to be classified simultaneously as being a type 1 and type 2mutation. Insofar as naturally occurring, archaebacterialproton-translocating retinal proteins exhibit a photocycle which isretarded as compared with that of the best-known proton-translocatingretinal protein, i.e. bacteriorhodopsin derived from Halobacteriumsalinarum (SEQ ID No. 1), and their all-trans retinal contents in thelight-adapted and dark-adapted states do not differ from each other bymore than 10%, they are likewise regarded as being muteins within thecontext of the present invention.

[0016] The term “homolog”, which is known to the skilled person, denotesa relationship between two or more peptides, polypeptides or proteinswhich can be determined, on the basis of the degree of congruencebetween the sequences, using known methods, for examplecomputer-assisted sequence comparisons (basic local alignment searchtool, S. F. Altschul et al., J. Mol. Biol. 215 (1990), 403-410). Thepercentage identity is calculated from the percentage of identicalregions in two or more sequences while taking into account gaps or otherspecial sequence features. As a rule, use is made of special computerprograms which employ algorithms which take the particular requirementsinto account.

[0017] Preferred methods for determining homology initially generate thegreatest degree of congruence between the sequences being investigated.Computer programs for determining the identity between two sequencesinclude, but are not restricted to, the GCG program package, includingGAP (Devereux, J., et al., Nucleic Acids Research 12 (12):387 (1984);Genetics Computer Group University of Wisconsin, Madison, (Wis.));BLASTP, BLASTN and FASTA (Altschul, S. et al., J. Mol. Biol. 215:403-410) (1999)). The BLASTX program can be obtained from the NationalCentre for Biotechnology Information (NCBI) and from other sources(BLAST Manual, Altschul S., et al., NCB NLM NIH Bethesda MD 20894;Altschul S., et al., Mol. Biol. 215: 403-410 (1990)). The well-knownSmith-Waterman algorithm can also be used for determining the percentageidentity.

[0018] Preferred parameters for the amino acid sequence comparisoncomprise the following: Algorithm: Needleman and Wunsch, J. Mol. Biol.48: 443-453 (1970) Comparison matrix: BLOSUM 62 from Henikoff andHenikoff, PNAS USA 89 (1992), 10915-10919 Gap penalty: 12 Gap lengthpenalty:  4 Threshold of similarity:  0

[0019] The GAP program is also suitable for being used with theabovementioned parameters. The abovementioned parameters are the defaultparameters for amino acid sequence comparisons in which gaps at the endsdo not alter the value. The invention therefore also encompasses fusionproteins, i.e. proton-translocating retinal proteins which possess afusion protein moiety. When sequences which are very short as comparedwith the reference sequence are being dealt with, it can furthermore benecessary to increase the expectation value up to a maximum of 100 000and, where appropriate, to decrease the word size down to a minimum of2.

[0020] It is possible to use other exemplary algorithms, gap openingpenalties, gap extension penalties and comparison matrices, includingthose mentioned in the program manual, Wisconsin package, Version 9,September 1997. The choice will depend on the comparison to be performedand, in addition, whether the comparison is carried out between sequencepairs, in which case GAP or best fit is preferred, or between a sequenceand an extensive sequence database, in which case FASTA or BLAST ispreferred.

[0021] Within the context of this application, a congruence of 40%, asdetermined using the abovementioned algorithm, is described as being 40%identity. The same applies, in a corresponding manner, to higherpercentages.

[0022] It has now been found, surprisingly, that combining a type 1mutation and a type 2 mutation in the proton-translocating retinalprotein according to the invention can lead to an extensive degree ofconstancy in the all-trans retinal content and to a retardation of thephotocycle. As a rule, the all-trans retinal content of the dark-adaptedform of the proton-translocating retinal protein according to theinvention does not differ from that of the light-adapted form by morethan 10%. In preferred embodiments, the differences are even smaller andare maximally 8 and even maximally 5%. It has furthermore been found,surprisingly, that it is precisely muteins whose all-trans retinalcontents in the light-adapted and dark-adapted states do not differsignificantly, i.e. by not more than 10%, which exhibit all-transretinal contents of at least 60% in both the light-adapted anddark-adapted states. This unexpected side effect is extremelyadvantageous since any increase in the proportion of molecules whichparticipate in the “trans cycle” leads, on illumination, to a moredistinct color change and consequently to an improvement in the opticalproperties.

[0023] The invention provides muteins which exhibit a constant all-transretinal content in the range from at least 60 to 100%, preferably from62% or 65% to 100%. Whereas most retinal proteins exhibit an all-transretinal content in the range from 60 or 65% to 85%, particularlypreferred embodiments provide for an all-trans retinal content of atleast 70% or 75%, at best even from 80% to 100%.

[0024] In a preferred embodiment, the natural proton-translocatingretinal protein whose mutein(s) exhibit(s) the abovementioned propertiesis an archaebacterial bacteriorhodopsin, e.g. a halobacterial rhodopsin,preferably Halobacterium salinarum bacteriorhodopsin (SEQ ID No. 1).

[0025] The protein-translocating retinal protein according to theinvention encompasses muteins which possess a type 1 mutation and a type2 mutation and whose amino acid sequence exhibits an identity of atleast 40% with the amino acid sequence SEQ ID No. 1. In otherembodiments, the identity is at least 50, 60 or 70%. In particularlypreferred embodiments, the identity is at least 80, 90 or 95%.

[0026] In another preferred embodiment, the proton-translocating retinalprotein is a homolog having an amino acid sequence which, in the regionof the C helix and/or the F helix, exhibits an identity of at least 60%with the corresponding amino acid sequences of the maturebacteriorhodopsin from SEQ ID No. 1. In other preferred embodiments, thepercentage identity in the case of the C helix and/or the F helix is atleast 70, 80 or 90%, preferably at least 95%. In this connection, thedegree of identity in the case of C helix and the F helix can be thesame or different.

[0027] The photocycle of the proton-translocating retinal proteinaccording to the invention is retarded by the type 1 mutation. In everycase, the thermal cycle time is more than 10 ms, preferably more than 1s or even more than 10 s. In particularly preferred embodiments, thethermal cycle time is in the region of minutes, i.e. it is more than 1min and, in other preferred embodiments, even more than 5 or 10 min.While, in the extreme case, the thermal cycle time can be up to 2 hours,it is as a rule not more than 90 or 60 min.

[0028] The type 1 mutation of the proton-translocating retinal proteincan consist of an amino acid substitution at one or more of the aminoacid positions which are involved in the catalytic cycle in the naturalprotein. The present invention therefore encompasses the retinalproteins in which, for example, one or more of the positions of theamino acids which are involved in the proton translocation, i.e. fromthe group of the amino acid residues D38, R82, D85, D96, D102, D104,E194 and/or E204 as depicted in SEQ ID No. 1, or the amino acid residueswhich correspond to them in homologous proteins, is/are altered. Apreferred amino acid substitution is D96N, i.e. the amino acid D atposition 96 of SEQ ID No. 1, or the corresponding amino acid in ahomologous protein, is replaced with N. Other preferred amino acidsubstitutions are D38R and D102R and/or D104R.

[0029] The type 2 mutation of the retinal proteins of the retinalproteins according to the invention leads to an amino acid substitutionat one or more of the amino acid positions which form the retinalbinding pocket and/or immediately adjacent positions. The retinalbinding pocket is understood as meaning the sum of the amino acids whichconfer on the Schiff's base of the retinal its characteristic chemicaland physical properties. The amino acids which form the retinal bindingpocket, or immediately adjacent amino acids, are selected from the groupof the amino acid residues Val49, Ala53, L93, Met118, Gly122, S141 andMet145 as depicted in SEQ ID No. 1 or the amino acid residues whichcorrespond to them in homologous proteins. In preferred embodiments, thetype 2 mutation is V49A, V49G, V49F, L93A, G122K, G122C, G122M, S141A,S141M, M145I, M145F, M145W, M145C or M145K. Surprisingly, these type 2mutations bring about a constancy in the proportion of all-trans in theretinylidene moiety of the bacteriorhodopsin in the light-adapted anddark-adapted states.

[0030] Absorption maxima and retinal isomer ratios of the Halobacteriumsalinarum wild type (WT), of the pure type 1 mutant D96N, of the puretype 2 mutants M145F and L93A and of the retinal proteins according tothe invention, i.e. D96N-M145F and D96N-L93A, are shown in the followingtable: Isomers λ_(max) in nm DA LA Strain DA LA all-trans 13-cisall-trans 13-cis WT 560 568 60 40 98 2 D96N 560 569 54 46 96 4 M145F 558559 72 28 86 14 D96N-M145F 560 560 66 34 67 33 L93A 541 541 80 20 82 18D96N-L93A 544 544 80 20 81 19

[0031] Examples of particularly preferred combinations of type 1 andtype 2 mutations are V49A-D96N, L93A-D96N and M145F-D96N (see FIG. 1).

[0032] In another embodiment, the retinal proteins according to theinvention are present in membrane-bound form, with the membrane having adensity of between 1.10 and 1.20 g/cm³. In a preferred embodiment, thedensity is between 1.175 and 1.185 g/cm³. Particular preference is givento the form of a purple membrane having a density of 1.18 g/cm³.

[0033] The invention furthermore provides nucleic acids which encode theabove-described retinal proteins. These nucleic acids can be produced,on the one hand, by mutating previously known genes forproton-translocating retinal proteins, e.g. the gene encoding theHalobacterium salinarum bacteriorhodopsin (Dunn et al., Proc. Natl.Acad. Sci. USA 78, 6744-6748, 1981), or, on the other hand, be preparedentirely synthetically using known methods. Since the genetic code ofthe Archaea does not differ from that of the Prokarya and Eukarya, it isalso possible to use the known rules to prepare nucleic acids which areintended to be transformed into halobacteria. However, the skilledperson will endeavor to allow for a codon usage which is matched to thehost organism which is in each case designated for the purpose. Thenucleic acids according to the invention can be ribonucleic acids and/ordeoxyribonucleic acids.

[0034] The invention furthermore provides vectors which comprise nucleicacids which encode the proton-translocating retinal proteins. Dependingon the host organism which is designated for this vector, the skilledperson will choose between archaebacterial vectors and vectors forexpression in Prokarya (E. coli, Bacillus, Pseudomonas, Klebsiella,etc.) or Eukarya (yeast, animal cell cultures (CHO, HeLa, COS, etc.),plants or plant cells and insect cells).

[0035] The invention furthermore provides a host cell which containseither a nucleic acid encoding a protein-translocating retinal proteinaccording to the invention or a vector according to the invention. Thehost cells are, for example, Archaea, preferably halobacteria,particularly preferably Halobacterium salinarum, whose transformationhas been described (Cline et al., Can. J. Microbial. 35, 148-152, 1989).Alternatively, it is also possible to express vectors according to theinvention in E. coli or other prokaryotic hosts and, if required, in theabovementioned eukaryotic cells as well.

[0036] The invention furthermore provides a photochromic compositionwhich can comprise, in addition to a proton-translocating retinalprotein according to the invention, stabilizers, foamformation-diminishing and/or UV light-absorbing additives and/orbuffering substances.

[0037] The photochromic composition according to the invention cancomprise, for example, glycerol, organic polymers and/or organicsolvents.

[0038] The proton-translocating retinal proteins according to theinvention, or the photochromic compositions which comprise them, can beused for producing optical films. The production of optical films frombacteriorhodopsins is already known and is described in detail, forexample, in Hampp et al., SPIE 3623, 243, 1999. An optical film whichhas been produced using the proton-translocating retinal proteinsaccording to the invention is suitable for optical recording. Anotherpossibility of using such optical films is in interferometry or inholographic pattern recognition. It is also possible to use opticalfilms as optical light modulators. In addition to this, it is possibleto conceive of producing capacious stores for optical data storage(Birge, Scientific American, 1995, 66).

[0039] The present invention furthermore provides the use of aproton-translocating retinal protein and/or a photochromic compositionas a security dye. In a first embodiment, the retinal protein and/or thephotochromic composition is/are applied to a document which requiressecurity or to an article which requires security. In anotherembodiment, the photochromic composition according to the invention, orthe retinal protein, which is applied to the document which requiressecurity or to the article which requires security can be fixed on thedocument or the article.

[0040] In this connection, the fixing according to the present inventioncan be affected by physical inclusion or covalent coupling to thedocument. According to the invention, the document which requiressecurity is, for example, a security paper, a pass or a banknote.However, according to the invention, the document can also be any otherdocument which requires security.

[0041] Finally, the present invention provides a process for producingdocuments which possess security features, which is characterized inthat, before, during or after the production of a document in acustomary manner, a proton-translocating retinal protein according tothe invention or a photochromic composition according to the inventionis applied and, where appropriate, fixed to it.

[0042] The following figures and examples explain the invention.

[0043]FIG. 1 shows the absorption spectra of differentproton-translocating retinal proteins according to the invention ascompared with the spectra of the wild type or of muteins which possessonly one mutation. In each case, the continuous lines show theabsorption spectrum in the light-adapted state while the dotted linesshow the absorption spectrum in the dark-adapted state. In detail:

[0044]FIG. 1A shows Halobacterium salinarum wild type having the aminoacid sequence depicted in SEQ ID No. 1. The absorption maximum is 568 nmin the light-adapted state and 560 nm in the dark-adapted state.

[0045]FIG. 1B shows the absorption spectrum of the type 1 mutant D96N.The absorption maximum shifts from 569 nm in the light-adapted state to560 nm in the dark-adapted state.

[0046]FIG. 1C shows the type 2 mutant V49A. The absorption maximum is549 nm in both the light-adapted and the dark-adapted states.

[0047]FIG. 1D shows the retinal protein according to the inventionhaving the double mutation V49A-D96N. The absorption maximum of 559 nmin the light-adapted state is displaced by 2 nm, to 557 nm, in thedark-adapted state.

[0048]FIG. 1E shows the type 1 mutation L93A. The absorption maximum is541 nm in both the light-adapted state and the dark-adapted state.

[0049]FIG. 1F shows the retinal protein according to the inventionhaving the double mutation L93A-D96N. The absorption maximum is 544 nmin both the light-adapted and dark-adapted states. In the dark-adaptedstate, this double mutant achieves an all-trans content of more than80%.

[0050]FIG. 1G shows the type 2 mutation M145F. The absorption maximum is559 nm in the light-adapted state and 558 nm in the dark-adapted state.

[0051]FIG. 1H shows the retinal protein according to the inventionhaving the double mutation D96N-M145F. In the dark-adapted state, thisdouble mutant achieves an all-trans content of 66%; the absorptionmaximum is 560 nm for both the light-adapted and the dark-adaptedretinal protein.

EXAMPLE 1

[0052] Producing a Photochromic Composition

[0053] In order to produce a photochromic composition, the individualproton-translocating retinal proteins are firstly isolated, either fromnatural halobacterium populations or recombinantly by transformingHalobacterium salinarum (Cline & Doolittle, J. Bact. 169, 1341-1344,1987) following site-specific mutagenesis of the bacteriorhodopsin gene(Dunn et al., Proc. Natl. Acad. Sc., USA 78, 6744-6748). Known methodsare used to isolate and purify the purple membrane of the transformedhalobacteria (Oesterhelt & Stoeckenius, Meth. Enzym. 31, 667-678, 1974).The method described in German patent application 199 45 798.0 can beused for isolating bacteriorhodopsin on a large scale.

EXAMPLE 2

[0054] Fixing the Photochromic Composition on a Surface

[0055] The photochromic composition according to the invention can, forexample, be incorporated physically into a matrix material.Specifically, 10 mg of purple membrane, containingbacteriorhodopsin-D96N/M145F, for example, are suspended uniformly in 4ml of a UV-curing dye (IFS 3000, from Schmitt) and applied to thedocument which requires security. After the document which has beenlabeled in this way has been subjected to UV illumination (in accordancewith the manufacturer's instructions), the purple membrane particles arelocated in the cured plastic.

EXAMPLE 3

[0056] Using Various Methods to Apply the Photochromic Composition

[0057] Screen Printing

[0058] The principle of screen printing is porous printing, in a similarmanner to a stenciling technique. The printing block consists of ascreen fabric which is provided with a dye-impermeable barrier layer.The printing motif can be chosen as desired. The printing is effected bythe dye-filled screen being scraped off using a doctor blade. Inconnection with this, the dye is transferred to the underlyingsubstrate. In order to prepare a screen printing dye, 100 mg of purplemembrane/ml are stirred overnight into a 7.2% solution of PVA (Mowioltype 56-98). When the rheological properties correspond to those of astandard sample, the mixture which is obtained can be printed using aconventional screen printing machine.

[0059] Offset Printing

[0060] 1 mg of purple membrane is stirred, at 50° C., into 5 ml of a dyewithout pigment (from Schmitt, UFO1). The mixture which is obtained inthis way can be printed using a conventional offset technique.

EXAMPLE 4

[0061] Producing an Abrasion-Resistant Security Feature

[0062] The photochromic composition can be made abrasion-resistant by,for example, using a hot-laminating appliance (GPM, Mylam 9) to laminatethe proton-translocating retinal protein-coated documents in a filmpocket of the GHQ-120TR type at a temperature of from 90 to 140° C.

EXAMPLE 5

[0063] Increasing the UV resistance of the security feature In order toincrease the UV resistance of the security feature according to theinvention, a UV absorber, or a derivative thereof, is added to thephotochromic composition at a concentration of from 1 to 30%, preferablyof from 3 to 10%, w/w. Preferred UV absorbers are benzophenone,hydroxynaphthoquinone, phenylbenzoxazole, cinnamic esters, sulfonamideand aminobenzoic esters.

1 1 1 248 PRT Halobacterium salinarum 1 Glu Ala Gln Ile Thr Gly Arg ProGlu Trp Ile Trp Leu Ala Leu Gly 1 5 10 15 Thr Ala Leu Met Gly Leu GlyThr Leu Tyr Phe Leu Val Lys Gly Met 20 25 30 Gly Val Ser Asp Pro Asp AlaLys Lys Phe Tyr Ala Ile Thr Thr Leu 35 40 45 Val Pro Ala Ile Ala Phe ThrMet Tyr Leu Ser Met Leu Leu Gly Tyr 50 55 60 Gly Leu Thr Met Val Pro PheGly Gly Glu Gln Asn Pro Ile Tyr Trp 65 70 75 80 Ala Arg Tyr Ala Asp TrpLeu Phe Thr Thr Pro Leu Leu Leu Leu Asp 85 90 95 Leu Ala Leu Leu Val AspAla Asp Gln Gly Thr Ile Leu Ala Leu Val 100 105 110 Gly Ala Asp Gly IleMet Ile Gly Thr Gly Leu Val Gly Ala Leu Thr 115 120 125 Lys Val Tyr SerTyr Arg Phe Val Trp Trp Ala Ile Ser Thr Ala Ala 130 135 140 Met Leu TyrIle Leu Tyr Val Leu Phe Phe Gly Phe Thr Ser Lys Ala 145 150 155 160 GluSer Met Arg Pro Glu Val Ala Ser Thr Phe Lys Val Leu Arg Asn 165 170 175Val Thr Val Val Leu Trp Ser Ala Tyr Pro Val Val Trp Leu Ile Gly 180 185190 Ser Glu Gly Ala Gly Ile Val Pro Leu Asn Ile Glu Thr Leu Leu Phe 195200 205 Met Val Leu Asp Val Ser Ala Lys Val Gly Phe Gly Leu Ile Leu Leu210 215 220 Arg Ser Arg Ala Ile Phe Gly Glu Ala Glu Ala Pro Glu Pro SerAla 225 230 235 240 Gly Asp Gly Ala Ala Ala Thr Ser 245

1. A proton-translocating retinal protein which is selected from thegroup comprising: (i) muteins of a natural proton-translocating retinalprotein derived from halophilic archaebacteria which exhibit a retardedphotocycle (type 1 mutation) and whose all-trans retinal contents in thelight-adapted and dark-adapted states do not differ from each other bymore than 10% (type 2 mutation) and/or (ii) homologs of the muteins (i)which possess a retarded photocycle and whose all-trans retinal contentsin the light-adapted and dark-adapted states do not differ from eachother by more than 10%.
 2. The proton-translocating retinal protein asclaimed in claim 1, characterized in that its retinal isomer compositionexhibits at least 60% all-trans retinal in both the light-adapted anddark-adapted states.
 3. The proton-translocating retinal protein asclaimed in claim 1 or 2, characterized in that the naturalproton-translocating retinal protein is an archaebacterial rhodopsin. 4.The proton-translocating retinal protein as claimed in one of claims 1to 3, characterized in that the archaebacterial rhodopsin is a rhodopsinderived from halobacteria.
 5. The proton-translocating retinal proteinas claimed in one of claims 1 to 4, characterized in that thearchaebacterial rhodopsin is Halobacterium salinarum bacteriorhodopsin(SEQ ID No. 1).
 6. The proton-translocating retinal protein as claimedin one of claims 1 to 5, characterized in that the homolog exhibits anamino acid sequence which exhibits at least 40% identity with the aminoacid sequence SEQ ID No.
 1. 7. The proton-translocating retinal proteinas claimed in one of claims 1 to 6, characterized in that the homologexhibits an amino acid sequence which, in the region of C helix and/orthe F helix, exhibits at least 60% identity with the amino acid sequenceSEQ ID No.
 1. 8. The proton-translocating retinal protein as claimed inat least one of claims 1 to 7, characterized in that the photocycle ofthe muteins containing a type 1 mutation exhibits a thermal cycle timeof more than 10 ms.
 9. The proton-translocating retinal protein asclaimed in at least one of claims 1 to 8, characterized in that the type1 mutation is an amino acid substitution at one or more of the aminoacid positions which, in the natural protein, are involved in thecatalytic cycle.
 10. The proton-translocating retinal protein as claimedin claim 9, characterized in that the amino acids which are involved inthe catalytic cycle are selected from the group comprising the aminoacid residues D38, R82, D85, D96, D102, D104, E194 and/or E204.
 11. Theproton-translocating retinal protein as claimed in at least one ofclaims 1 to 10, characterized in that the type 2 mutation is an aminoacid substitution at one or more of the amino acid positions which formthe retinal-binding pocket.
 12. The proton-translocating retinal proteinas claimed in claim 11, characterized in that the amino acids formingthe retinal-binding pockets are selected from the group comprising theamino acid residues Va149, Ala53, L93, Met118, Gly122, S141 and Met145.13. The proton-translocating retinal protein as claimed in one of claims1 to 12, characterized in that it is a mutein of the Halobacteriumsalinarum bacteriorhodopsin (SEQ ID No. 1) containing mutations selectedfrom the following group: V49A-D96N; L93A-D96N; M145F-D96N or a homologof such a mutein.
 14. The proton-translocating retinal protein asclaimed in at least one of claims 1 to 13, characterized in that theretinal protein is present in the form of a purple membrane.
 15. Theproton-translocating retinal protein as claimed in claim 14,characterized in that the density of the purple membrane is between 1.10and 1.20 g/cm³.
 16. The proton-translocating retinal protein as claimedin claim 15, characterized in that the density of the purple membrane isfrom 1.175 to 1.185 g/cm³.
 17. A nucleic acid which encodes aproton-translocating retinal protein as claimed in one of claims 1 to13.
 18. A vector which comprises a nucleic acid as claimed in claim 17.19. A host cell which contains a nucleic acid as claimed in claim 17and/or a vector as claimed in claim
 18. 20. A photochromic compositionwhich comprises at least one proton-translocating retinal protein asclaimed in one of claims 1 to 16 and which furthermore comprises one ormore additives which are selected from stabilizers, foamformation-diminishing additives, UV light-absorbing additives andbuffering substances.
 21. The photochromic composition as claimed inclaim 20, characterized in that it comprises glycerol, organic polymersand/or organic solvents.
 22. A use of a proton-translocating retinalprotein as claimed in at least one of claims 1 to 16 and/or aphotochromic composition as claimed in claim 20 or 21 for producingoptical films.
 23. The use as claimed in claim 22, characterized in thatthe optical film is suitable for optical recording.
 24. The use asclaimed in claim 22, characterized in that the optical film is suitablefor interferometry.
 25. The use as claimed in claim 22, characterized inthat the optical film is suitable for holographic pattern recognition.26. The use as claimed in claim 22, characterized in that the opticalfilm is suitable as an optical light modulator.
 27. The use of aproton-translocating retinal protein as claimed in one of claims 1 to 16for producing capacious stores for optical data storage.
 28. The use ofa proton-translocating retinal protein as claimed in at least one ofclaims 1 to 16 or of a photochromic composition as claimed in at leastone of claims 20 and 21 as a security dye.
 29. The use as claimed inclaim 28, characterized in that the proton-translocating retinal proteinand/or the photochromic composition is/are applied to a document whichrequires security or to an article which requires security.
 30. The useas claimed in claim 29, characterized in that the proton-translocatingretinal protein and/or the photochromic composition which is/are appliedto the document or article which requires security is/are fixed on thedocument or the article.
 31. The use as claimed in claim 30,characterized in that the fixing is effected by physical inclusion orcovalent coupling to the document or the article.
 32. The use as claimedin one of claims 29 to 31, characterized in that the document whichrequires security is a security paper.
 33. The use as claimed in one ofclaims 29 to 31, characterized in that the document which requiressecurity is a pass.
 34. The use as claimed in one of claims 29 to 31,characterized in that the document which requires security is abanknote.
 35. A process for producing documents possessing safetyfeatures, characterized in that, before, during or after the productionof a document in a customary manner, a proton-translocating retinalprotein as claimed in one of claims 1 to 16 or a photochromiccomposition as claimed in claim 17 or 18 is applied and, whereappropriate, fixed to it.